Staphylococcus aureus is a major human pathogen that causes significant morbidity and mortality in both hospital- and community-acquired infections. The appearance of multidrug-resistant strains has compounded this problem, galvanizing efforts to identify novel therapeutic targets. Extremely attractive targets are bacterial iron acquisition systems, which are vital to infection by many pathogens. In this regard, we have found that efficient procurement of heme-iron is required for S. aureus pathogenesis and disease. Heme-iron is primarily associated with hemoglobin (Hb), the most abundant source of iron in the human host. We have identified two integral components of the staphylococcal machinery that mediates heme-iron acquisition: the iron-regulated surface determinant system (Isd) and the heme transport system (Hts). Based on these recent discoveries, new studies are proposed to understand the mechanism and function of Isd/Hts in heme-iron acquisition from Hb and in the pathogenesis of S. aureus infection. Preliminary experiments suggest that this mechanism involves Hb recognition and heme-iron removal by a cell wall-anchored protein (IsdB), heme-iron transport through cytoplasmic membrane transport systems (IsdDEF and HtsABC), and cytoplasmic heme-iron degradation by monooxygenases (IsdGI). In turn, iron derived from heme is released for use as a nutrient source that is essential for S. aureus disease progression. This proposal focuses on testing this model for Hb-iron acquisition. We will utilize genetics, inorganic chemistry, biochemistry, and animal infection experiments to (i) define the functional consequence of the interaction between Hb and IsdB, (ii) delineate the role of membrane transporters in heme-iron transit into the cytoplasm, and (iii) determine the contribution of cytoplasmic heme degradation to S. aureus pathogenesis. Results from these studies will yield a molecular blueprint of the heme-iron acquisition machinery in S. aureus, and permit the rational design of small molecule inhibitors for therapeutic intervention in S. aureus infection. Furthermore, the conservation of the Isd/Hts systems in the microbial agents that cause anthrax, tetanus, and listeriosis will enable our results to be extrapolated to multiple humanpathogens.